The long-term goals of this project are to understand the mechanisms of cellular repair of DNA damage after ionizing radiation, and to identify and study the genes that control this repair. The research uses the budding yeast Saccharomyces cerevisiae as a model organism. Most of the repair pathways and many of the genes that act in yeast DNA repair are conserved between yeast and mammals. Therefore, by using the genetic and molecular methods available for yeast, we can better understand repair in humans. DNA repair mechanisms play a central role in maintaining life and health. Mutations in many human repair genes initially identified by homology to yeast genes are now known to confer specific diseases, and effective DNA repair is essential for maintaining the stability of the genome in both somatic and germ-line cells. This is crucial for the avoidance of germ-line mutations and somatic cell changes that can lead to malignant transformation, and for preventing cellular lethality and premature aging. In yeast, mutant strains separately deleted for almost every gene in the genome have recently become available. In collaboration with the laboratory of Dr. Martin Brown at Stanford, the mutants are being used in this project to identify all the genes that can mutate to confer radiation sensitivity in yeast. Double mutant analysis and research on the phenotypes conferred by recently identified mutants will be used to understand repair pathways and build on existing knowledge. Genes of current focus in the project are RAD61 and MDM20. A molecular focus will complement the genetic approach. This will use methods developed here to measure double-strand DNA breaks (DSBs) very sensitively with pulsed-field gel electrophoresis of yeast chromosomes. Yeast strains with a circular chromosome homologue provide special value, because a single DSB anywhere along this molecule yields a linear derivative of a uniform size, which can be visualized as a new band on a gel. The slightly larger existing linear homologue from a heterozygous diploid provides a control value for the fraction of unbroken molecules in the same lane. DSBs are known to be lethal in repair-deficient yeast mutants, and the PFGE system is being used here to determine the true role of DSBs in causing lethality in wild-type cells. Recent and existing mutants will be studied in detail with this system to determine the molecular involvement of each gene in DSB repair, sister chromatid exchange and recombination between chromosomes.